Tolerance Potentials of Cocoa (Theobroma cacao) in Hydrocarbon Polluted Soils

In the present study the tolerance potentials of T. cacao in hydrocarbon polluted soil was evaluated. Top soil (0-25 cm depth) was collected from three points, bulked to form composite soil sample. Eight kilograms of the soil sample each were weighed into twenty five (25) perforated bags. The bags were polluted with 0 ml/kg, 50 ml/kg, 100 ml/kg, 150 ml/kg and 200 ml/kg of crude oil respectively with five replicates for each concentration and allowed for 2 weeks before planting. Three seeds of cocoa were sown in each of the polythene bags. Water was applied every three days to keep the soil moist. The results showed that cocoa plants at 8 weeks after planting (WAP) had significantly higher height (P<0.05) than those of 4 weeks after planting (WAP). The plant Original Research Article Agbor et al.; BBJ, 7(3): 111-121, 2015; Article no.BBJ.2015.052 112 height, petiole length, number of vine had no significant difference (P>0.05) at the different concentrations of crude oil. While the leaf length, leaf area, number of leaves, leaf width and vine length shows significant difference (P<0.05) and in treatment dependent manner. The results also showed that the concentrations of Cu, Ni, Cr and Zn in the polluted soils were treatment dose dependent but did not significantly affect the plant tolerance at P<0.05. The pH, phosphorus, nitrogen content, and other physicochemical properties of the soil were not significantly affected by the crude oil treatment. The T. cacao has been found to be tolerant to hydrocarbon polluted soil environment, its usage in oil spill impacted area should be encourage.


INTRODUCTION
Phytoremediation is a technique in environmental biotechnology that embraces the use of plants to remove, transfer, stabilize and destroy organic and inorganic contamination from land, groundwater and surface water. Phytoremediation is gaining research interest such that its involves the use of plants which are environmentally friendly in solving the problems of the environment. Phytoremediation offers an aesthetic and low-cost remediation technique to sites with low to moderate contaminant concentration where the pollutants are not located very deep. Crude oil is a raw material for the production of petroleum and other chemicals, and has quite been one of the most important energy sources in the world. It is a natural resource of the industrialized nations because it can generate heat, drive machinery, and fuel vehicle and airplane. Its components are used to manufacture almost all chemical products such as; plastics, detergents, paint and even medicine. However, pollution of soil and water environment by crude oil spill as a result of exploration, production, maintenance, transportation, storage and accidental discharge releases hazardous chemicals to the ecosystem. The presence of the hydrocarbons in terrestrial and aquatic environment affects the plants adversely by creating conditions which make essential nutrients like; nitrogen and oxygen needed for plants growth unavailable to them [1]. Oil contamination causes slow rate of germination in plants, [2] reported that this effect could be due to oil which acts as a physical barrier preventing or reducing access of seed to water and oxygen.
In today's industrial society, there is no way to avoid the exposure to toxic chemicals and metals because heavy metals are enriched in the environment by human activities of different kind like in developed countries, heavy metal pollution becomes serious due to mining, minerals, smelting and tannery industry [3]. From plants nutritional studies, it is known that plants require a certain amount of trace element, that they respond differently to enhanced or lowered trace element supply, and that in some cases agricultural products may be contaminated with toxic heavy metals. Metal concentrations in contaminated soil result in decreasing soil microbial activities soil fertility and yield losses [4]. Phytoremediation could be easily achieved with higher plants that can accumulate heavy metals at different pollution levels. Ogbo et al. [5] used Paspalum scrobiculatum L. in the phytoremediation of crude oil impacted soil and reported a significant reduction in the hydrocarbon content of the soil. Shahrzad et al. [6] reported on the phytoremediation potential of Trifolium resupinatum in the degradation of hydrocarbons in soil, they found a significant reduction in the total hydrocarbon content of the soil. Differences in metal accumulation may exist between and within plant population. This study examined the tolerance of cocoa plant in hydrocarbon polluted soils through the evaluation of the morphological features of the plant, physicochemical and heavy metal content of the soil.  (8 kg) of the soil sample each was weighed into twenty five (25) perforated bags, leaving a space of 2.5 cm from the top to make allowance for addition of water. The perforation of planting bags was at the base to ensure proper drainage and better aeration of soil. The 20 polythene bags were polluted with the following concentrations of crude oil: 0 ml/kg, 50 ml/kg, 100 ml/kg, 150 ml/kg, and 200 ml/kg, respectively and the setup were allowed for 2 weeks before planting.

Planting of Seeds and Water Application
Three (3) seeds of cocoa were sown in each polythene bag containing the polluted soil and that of the control, application of water was done also to keep the soil moist and to avoid drought. Monitoring of plants was done during the planting period to ensure healthy plants growth and the morphological attributes of the plants were taken. Data were taken four weeks after planting (4WAP) and eight weeks after planting (8WAP). Soil samples were taken at the end of the experiment for physicochemical and heavy metals analysis at 4WAP and 8WAP.

Physicochemical Analysis of Soil
The collected soil samples were sieve and air dried. Soil pH was determined using a hand held pH meter in distilled water according Thomas [7]. Soil organic carbon was determined by the chromic acid digestion method of Walkley and Black as reported by Helmke and Sparks [8].
The total (N) Bremner [9] available P was determined by Bray-P method as described by Kuo [10]. Exchangeable soil properties were extracted with neutral normal ammonium acetate buffer according to Helmke and Sparks [8].
Potassium and Sodium were determined using a Flame Photometer.

Heavy Metal Content of Soil
Two gram (2 g) of each of the sieved soil samples was weighed into a conical flask and digested with 10 ml of 50% hydrochloric acid on a hot plate until 2-3 ml of acid was left. Ten (10) ml de-ionized water was added to the content and demented into 50ml volumetric flask after additional water rinsing and decanted and made up to the mark with de-ionized water. A blank was prepared without soil sample. The extract solution was poured into polythene bottles from where each sample was analyzed for Cd, Cu, Fe, Pd, and Zn using Atomic Absorption Spectrophotometer Model 205.

Data Collection
The following parameters were taken; days of germination, plants heights, number of leaves, leaf length, leaf width, leaf area, numbers of branches, vine length, petiole length, numbers of vein.

Statistical Analysis
Data collected were subjected to a two-way analysis of variance (ANOVA), while significant mean were separated using least significant difference (LSD) Test at 5% probability level.

Morphological Features of T. cacao Grown on Hydrocarbon Impacted Soil
A phytoremediation plant is one that can absorb or extract, degrade contaminant, such plant should be able to tolerate high presence or concentrations of contaminant and then grow at a faster rate. This present study has shown that T. cacao is a good material for phytoremediation due to the high growth rate recorded. One main challenges that have hindered the full application of phytoremediation strategies in polluted environment is the stress that is often induce on the rate of seed germination and sprouting of plants. The stress imposed on the seed could be as results of heavy metal contents of the soil that distorted the emergence of the plants. The seed germination of T. cacao delayed may be due to the hydrocarbon contents of the soil (Plate 1).

Heavy Metal Properties of Crude oil Impacted Soil Grown with Cocoa
It is well known that elements such as Cu, Mo, Ni, Cr, and Zn among others are essential for plant growth at low concentration Taiz and Zeiger [13]. Blaylock and Huang [14] reported that these elements beyond certain threshold concentration become toxic to most plant species. Although, these elements were observed to be significantly high (P<0.05) in the soil at varying concentration of crude oil, the T. cacao was observed to be resistant to the effect of the heavy metals. The growth of T. cacao over a period of 8WAP indicates that the plant is also highly tolerant to chromium at a high concentration. Lead is a metal with limited availability for plant uptake due to complexities with solid soil fractions [15], sequential extractions, or a very small fraction of total soil. Lead was observed to be present in a form directly available to the plant. A number of studies have indicated the potential of phytoremediation in reducing the concentration of various contaminants including petroleum hydrocarbon [16]. Interestingly, the growth attributes of the plant remained unaffected even after 8WAP. Heavy metal content of the crude oil polluted soil grown with T. cacao. The result obtained for the heavy metal content of T. cacao soil shows that the iron (Fe) and manganese (Mn) present in the soil at 200 ml crude oil pollution was significantly higher (p<0.05) than that of soil polluted with 150 ml followed by control which was found to have significantly low (P <0.05) Fe and Mn content. The Nickel (Ni) content of the soil at 50 ml pollution level had no significant difference (P>0.05) with the control but significantly higher (P<0.05) than soil polluted with 100 ml/kg, followed by 150 ml/kg and 200 ml/kg that had no significant difference (p <0.05). The copper (Cu) content of the soil polluted with 50 ml, 100 ml, 150 ml/kg and 200ml/kg had no significant difference (p <0.05) in its mean values but significantly higher (p <0.05) than the mean of control (Table 3). The zinc (Zn) was observed to be significantly higher (p <0.05) in the leaves of soil polluted with 200 ml/kg, followed by soil polluted with 150 ml/kg and 100 ml/kg that had no significant difference (p <0.05) in its mean values. This was also followed by soil polluted with 50 ml/kg of crude oil while the control had significantly low (p <0.05) Zn content. The result obtained for the cadmium (Cd) content of the soil polluted with 200 ml/kg, 150 ml/kg and 100 ml/kg was significantly higher (p<0.05) than that obtained from the control and 50 ml/kg polluted soil with no significant difference (p<0.05). The chromium, (Cr) and vanadium (V) content of the soil had no significant difference (p<0.05) with the mean of the control. The lead (Pb) content of the soil polluted with 200 ml/kg was significantly higher (p <0.05) than that of soil polluted with 150 ml/kg, 100 ml/kg with no significant difference (p >0.05) in the mean. This was followed by soil polluted with 50 ml/kg while the control had significantly low (P <0.05) lead content. The Cobalt (Co) content of the soil in control was significantly higher (p <0.05) than that of soil polluted with 50 ml/kg followed by soil polluted with 100 ml/kg, 150 ml/kg, 200 ml/kg which had no significant difference (p >0.05) (Figs. 1-7

Soil Physicochemical Properties in Crude Oil Impacted Soil Grown with Cocoa
The result for soil moisture content shows that soil polluted with 50 ml of crude oil and the control had no significant difference (P>0.05) in their mean values but significantly higher (P<0.05) than the mean value obtained from soil polluted with 100 ml/kg, 150 ml/kg and 200 ml/kg of crude oil. The pH value of polluted soil at different pollution level had no significant difference (P>0.05) but significantly higher (P<0.05) than the control values (Table 3). It was observed from this result that the organic carbon content of the soil polluted with 200 ml/kg of crude oil was significantly higher (p<0.05) than soil polluted with 50ml/kg, 100ml/kg, and 150 ml/kg of crude oil which had no significant difference (P>0.05) but significantly higher (p<0.05) than the control. Stephen and Ijah [17] evaluated the potentials of Glycine max and Sida acute for phytoremediation of waste lubricating oil polluted soil and reported that the pH, moisture, electrical conductivity and phosphorus levels were lower in the S. acuta treatment than G.max treatment. This study revealed that the nitrogen content of the polluted soil and control had no significantly difference (P>0.05).
Observation also showed that the soil polluted with 100 ml/kg, 150 ml/kg and 200 ml/kg of crude oil had significantly high phosphorus content (P<0.05) and significantly higher (P<0.05) in soil polluted with 50 ml/kg crude oil and it control group. Stephen et al. [18] reported that no significant difference (p>00.5) exist in the pH, organic carbon and organic matter content while the moisture and phosphorus concentration of the polluted soil was significantly different (p>00.5). The potassium level in the control was significantly higher (P<0.05) than that of soil polluted with 50 ml/kg, 100 ml/kg and 150 ml/kg of crude oil while 200 ml/kg polluted soil had the lowest potassium content in the soil. It was observed that the magnesium content of soil polluted with 200 ml/kg, 150 ml/kg, 100 ml/kg of crude oil had no significantly difference (P>0.05) but significantly higher (P<0.05) than soil polluted with 50 ml/kg and control with no significant difference (P>0.05). The calcium content of the soil polluted with 200 ml/kg and 150 ml/kg of crude oil had no significant difference (P>0.05) but significantly higher (P<0.05) than soil polluted with 100 ml/kg, followed by the soil polluted with 50 ml/kg of crude oil. The control was observed to have the lowest calcium content. The hydrogen and aluminum (H + and Al 3+ ) and base saturation of the polluted and unpolluted soil were not significantly different (P>0.05). The sodium and effective cation exchange capacity (ECEC) content of the soil polluted with crude oil were significantly higher (P<0.05) than the control.

CONCLUSION
The T. cacao exhibited a high tolerance to crude oil polluted soils. However, this plant is worthy for further studies with respect to its use in phytoremediation of crude oil polluted soil because of its availability and abundance in Nigeria.